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Norfloxacin (NOR) in milk may influence mammalian cell replication and bring about a decrease in the efficiency for treating infection in humans. However, current techniques for detecting NOR usually require expensive instruments and trained personnel. In this work, we have developed a low-cost and simple method via paper-based fluorescent immunoassay for highly sensitive and selective detection of NOR in milk at picogram level. The NOR monoclonal antibody labeled with quantum dots is used as a detection probe to recognize the corresponding NOR, which can quantitatively detect NOR on paper-based devices. The detection limits in aqueous solution and milk are 1 pg/mL and 10 pg/mL, respectively. The developed paper-based method provides a cheap, sensitive, eco-friendly, and rapid approach for quantitative detection of trace NOR in milk, which may find wide applications in food safety inspection. Noteworthy, the method is especially suitable for applications at resource-limited and on-site settings.

Both the ABO and Rhesus (Rh) blood groups play crucial roles in blood transfusion medicine. Herein, we report a simple and low-cost paper-based analytical device (PAD) for phenotyping red blood cell (RBC) antigens. Using this Rh typing format, 5 Rh antigens on RBCs can be simultaneously detected and macroscopically visualized within 12 min. The proposed Rh phenotyping relies on the presence or absence of hemagglutination in the sample zones after immobilizing the antibodies targeting each Rh antigen. The PAD was optimized in terms of filter paper type, antibodies, and distance of the visualization zone. In this study, the optimal conditions were Whatman filter paper Grade 4; anti-D, -C, -E, -c, and -e antibodies; RBC suspension of 30%; and a visualization zone of 1 cm above the sample zone. The accuracy of simultaneously phenotyping the five Rh RBC antigens in the blood samples (n = 4692) was 99.19%, comparable with the accuracy of the gold-standard tube method used by blood bank laboratories in several regions of Thailand. Furthermore, decision making based on this method can be assisted by deep learning. After implementing a two-stage objective detection algorithm (YOLO v4-tiny) and classification model (DenseNet-201), the ambiguous images (n = 48) were interpreted with 100% accuracy. The PAD integrated with customized-region convolutional neural networks can reduce the interpretation discrepancies in RBC antigen phenotyping in any laboratory.

Brentuximab vedotin (BV) is approved for the treatment of patients with relapsed or refractory CD30-positive Hodgkin lymphoma, and relapsed or refractory systemic anaplastic large-cell lymphoma. Several uncertainties remain regarding the optimal use of the drug in its approved indications as well as outside them. This article reports recommendations on the use of BV issued during a consensus project, sponsored by the Italian Society of Hematology (SIE) and its affiliate societies, Società Italiana di Ematologia Sperimentale (SIES) and Gruppo Italiano Trapianto di Midollo Osseo (GITMO). Scientific evidence on BV was evaluated by a panel of experts, and consensus was developed by group discussion for key questions selected according to the clinical relevance. The following key issues were addressed: testing CD30 positivity to assess eligibility to BV; assessing practice indications of BV in Hodgkin lymphoma and systemic anaplastic large-cell lymphoma; providing pretreatment evaluation of patients candidates to BV; monitoring the response to BV; managing patients treated with BV; and assessing the role of BV in other CD30-positive lymphomas.

Branched zinc oxide nanorods (BZR) arrays, an array with high charge carries collection efficiency and specific surface area, are grown on the reduced graphene oxide-paper working electrode for the first time to construct a paper-based electrochemical (EC) immunosensor. Typically, the BZR are fabricated via a simple hydrothermal process, which can provide abundant sites for antibodies loading. By combining the large surface area of porous zinc oxide (PZS) and good biocompatibility of gold nanoparticles (AuNPs), PZS–AuNPs (PZS@Au) nanocomposites are designed to label horseradish peroxide (HRP) and antigens. After a competitive reaction between antigens and PZS@Au nanocomposites labeled antigens, the signal labels are introduced into the immunosensor, in which, HRP participate in biocatalytic precipitation process. The produced precipitate reduces the electrode surface area and hinders the electron transfer. With the increase of concentration of antigens, the signal labels introduced into the sensor decrease, thus, a signal-on immunoassay for α-fetoprotein detection is constructed. The proposed paper-based EC immunosensor combines enzymatic biocatalytic precipitation reaction and competitive immunoassay mode for the first time, and possesses a wide linear range from 0.2 pg mL −1 to 500 ng mL −1 with a detection limit of 0.08 pg mL −1 . In addition, the proposed method is simple, sensitive and specific and can be a promising platform for other protein detection.

An immunobinding assay is described that tests antigens attached to nitrocellulose paper against antisera contained in microtiter plates. In comparison to a conventional microtiter plate radioimmunoassay, the nitrocellulose paper radioimmunoassay is clearly superior in both antigen attachment and antibody binding. Studies using bovine serum albumin and human IgG demonstrated superior antigen attachment extending from 5-fold in a physiological solution, to 50-fold in 50% fetal calf serum, to over 1000-fold in detergent solutions. With titrations using a rabbit anti-human IgG serum, the antibody binding in the nitrocellulose paper radioimmunoassay averaged over 5 times the binding in the microtiter plate radioimmunoassay. The nitrocellulose paper radioimmunoassay was also modified to quantitate human IgG. With this assay, 15 pg of human IgG inhibited the antibody binding by 50%. The nitrocellulose paper radioimmunoassay is easy to perform, and, since it combines the antigen-binding properties of the nitrocellulose paper with the convenience of assaying samples in microtiter plates, this assay should prove useful for investigating the many antigens that attach to nitrocellulose paper.

A novel technique for establishing short term clones of antigen- or mitogen-activated splenic B lymphocytes is described. Spleen cells are plated onto the surface of filter paper discs and subsequently stimulated by antigen or mitogen in situ; activated B cells proliferate and differentiate into pure colonies of cells analogous to bacterial colonies growing on agar. These colonies of lymphocytes may be characterized in a series of replica hemolytic-plaque, autoradiographic, or immunoenzyme assays making possible a full characterization of the frequency of secreted idiotopes and paratopes and of the cells that produce them. Coloby induction by either antigen or mitogen occurs under idnentical conditions, thus a rigorous comparison between the mitoge-selected and antigen-selected antibody repertoires may be made.

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is known to inhibit matrix metalloproteinases, aggrecanases, and tumor necrosis factor (TNF)-alpha-converting enzyme (TACE, ADAM17). These metalloproteases participate in different aspects of joint destruction in inflammatory arthritis. To determine the relative importance of this inhibitor in joint pathology, wild-type and Timp3-/- mice were immunized with methylated bovine serum albumin followed by arthritis induction by intra-articular injection of the same antigen. Animals were monitored for up to 14 days after challenge, and joint tissues were analyzed by routine and Safranin O staining and for the presence of aggrecan neoepitopes produced by metalloprotease cleavage. Serum TNF-alpha was measured by immunoassay. Compared to wild-type animals, Timp3-/- mice showed a dramatic increase in the initial inflammatory response to intra-articular antigen injection, and serum TNF-alpha levels were greatly elevated in the Timp3-/- animals after immunization. However, these differences in clinical features disappeared by days 7 to 14. No difference in Safranin O staining or aggrecan cleavage site neoepitope abundance was seen. Thus, in inflammatory joint disease TIMP-3 likely dampens the inflammatory response of TNF-alpha by reducing ADAM17 activity.

A method is described for producing 1–3 μm sized particles of nitrocellulose (NC) which are able to absorb protein. Protein is absorbed onto preformed particles made by first dissolving a sheet of nitrocellulose paper in DMSO, and then precipitating it with sodium carbonate/bicarbonate buffer. The efficiency of binding is the same as that of an equivalent sheet of non-processed NC filter paper. Antibodies absorbed onto preformed particles are not exposed to DMSO and carbonate buffer and therefore retain a high antigen binding capacity. Antigen and antibody-absorbed NC preformed particles were used to capture antibody and antigen, respectively. Using lysis buffer, the captured antibodies and antigens were readily released from the NC particles. This makes the latter an appropriate matrix for immunoprecipitation assays either for an antigen or for specific antibody. Antigen-coated NC particles were specifically aggregated (‘agglutinated’) by specific antibodies and thus can be used in semi-quantitative tests.

The nematode intestine is continuous with the outside environment, making it easily accessible to anthelmintics for parasite control, but the development of new therapeutics is impeded by limited knowledge of nematode intestinal cell biology. We established the most comprehensive nematode intestinal functional database to date by generating transcriptional data from the dissected intestines of three parasitic nematodes spanning the phylum, and integrating the results with the whole proteomes of 10 nematodes (including 9 pathogens of humans or animals) and 3 host species and 2 outgroup species. We resolved 10,772 predicted nematode intestinal protein families (IntFams), and studied their presence and absence within the different lineages (births and deaths) among nematodes. Conserved intestinal cell functions representing ancestral functions of evolutionary importance were delineated, and molecular features useful for selective therapeutic targeting were identified. Molecular patterns conserved among IntFam proteins demonstrated large potential as therapeutic targets to inhibit intestinal cell functions with broad applications towards treatment and control of parasitic nematodes., Highlights • The intestine is an ideal target for therapeutics to control parasitic nematodes. • This is a pioneering pan-phylum study of the intestinal transcriptome/proteome in the Nematoda. • Conserved intestinal cell functions were delineated for the phylum clades and species of nematodes. • The possibility for selective targeting for therapeutic purposes is discussed. Using multi-omic data integration and evolutionary considerations, we derived a natural history of births, deaths and variation in family members for over 10,000 intestinal protein families conserved among ten nematode species. The development of therapeutics is explored in relation to conserved intestinal cell functions and molecular features specific to the parasite proteins.

This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of autoantibodies against the nuclear protein Scl-70. The isolation of Scl-70 from rat livers and the conditions for the ELISA are described. Compared with the already established double diffusion in gel for detection of anti-Scl-70 antibodies this ELISA has advantages.

A device is described which is used to measure the concentration of antigen in solution. A wedge-shaped gap containing the antigen to be detected is bounded on one side by a reservoir containing specific antiserum and on the opposite side by a detection slide which has been previously coated with a layer of adsorbed antigen. As the antibodies diffuse through the gap, they complex with the soluble antigen in the wedge until these become saturated leaving additional antibody free to complex with antigen on the detection slide. By allowing diffusion to continue for an appropriate time, it is possible to relate the distance along the gap where this has occurred to the concentration of antigen within the gap. Using human IgG as a model antigen, we have been able to measure concentrations down to approximately 10 μm/ml with this test.

We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.

Utilizing the principles of counterimmunoelectrophoresis, a technique was devised to immunologically identify non-denatured protein antigens resolved by polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred from polyacrylamide gel to antibody-containing agarose gel in a commercially available blotting apparatus. Detection of the antigen/antibody complex by Coomassie blue staining (in the ng range for unlabeled antigen) or autoradiography (in the pg range for radiolabeled antigen) established the sensitivity and specificity of this method. Similar sensitivity could be obtained following conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis after partial removal of the detergent and possible renaturation of proteins. Placement of a nitrocellulose sheet on the anodal side of the agarose gel produced a ‘negative replica’ (proteins/polypeptides not reacting with specific antibodies in the agarose gel). Counterimmunoblotting provides an additional method for molecular weight determination of protein antigens with sensitivity and specificity comparable to established nitrocellulose blotting (unlabeled proteins) or immunoprecipitation (labeled proteins) procedures.

The epidermolysis bullosa acquisita antigen is a major constituent of the basement membrane zone beneath stratified squamous epithelium. The antigen which is recognized in extracts of skin basement membrane by Western blot analysis with polyclonal or monoclonal antiepidermolysis bullosa acquisita antigen antibodies as 2 chains (a major chain of 290,000 and a minor chain of 145,000) has a native molecular weight over 800,000. Both epidermolysis bullosa acquisita antigen chains contain carbohydrate and the 290K chain is sensitive to collagenase.

The agent of measles (rubeola) has been assumed to be a paramyxovirus on the basis of its electron microscopic appearance, its size, its sensitivity to ether and detergents, and the presence of hemagglutinin in the viral envelope. Thus far, however, the one attribute common to myxoviruses, namely neuraminidase, has not been described for measles virus. We have demonstrated that specific neuraminidase activity is associated with measles viral hemagglutinin. The evidence suggests strongly that the enzyme is part of the viral envelope.

Three strains of the satellite (SV) of tobacco necrosis virus (TNV) were compared antigenically and in their ability to multiply with the help of different strains of TNV. Their antigenic specificity was not influenced by the strain of TNV but depended on the particular strain of SV used in the mixture. Antigenically the three strains were distinctly different, and this was reflected in the 49 changes in the amino acid composition of SV 1 and SV 2 . SV 1 and SV 2 multiplied well in mechanically inoculated leaves, whereas SV 3 did best in tobacco roots infected by the aid of the vector Olpidium brassicae . An ambient temperature of 36 °, which inhibited the multiplication of TNV, also affected SV. All strains crystallized readily and virus was lost in crystals during purification unless care was taken. From amino acid analysis and two-dimensional peptide maps, the molecular weight of the protein subunit of SV 1 was found to be 22,800 and to contain 208 amino acid residues. Of the 208 amino acid residues, 182 were accounted for in tryptic peptides separated by ion exchange and Sephadex chromatography. In the TNV and SV association, the RNA of SV codes for its coat protein. On the basis of the triplet code, the coding for the coat protein would require 624 of the 1200 nucleotides, and therefore SV can code for another protein. The help required by SV from TNV is discussed.

The basic protein encephalitogen, without, prior denaturation, was rapidly digested with such proteolytic enzymes as trypsin, pronase, chymotrypsin, and pepsin; from 80–90% of the proteolysis occurred within 1 hr. The number of peptides released by these enzymes after 2 hr were about 28, 54, 20, and 14, respectively. Each proteolytic digest was chromatographed on Sephadex G-25 and G-75 in order to separate the peptides from large partially digested material. Only the peptide fractions obtained from the pepsin digestion possessed significant activity in inducing experimental allergic encephalomyelitis (EAE). One of the peptide fractions had higher encephalitogenic activity, on a weight basis, than either the original protein or the unfractionated digest. In contrast, the chymotryptic digest, while lacking encephalitogenic activity, possessed antibody-combining activity as shown by the Ouchterlony and passive hemagglutination-inhibition (PHI) test. The pepsin digest produced peptides which gave a negative Ouchterlony test. The tryptic peptides were negative in all tests—Ouchterlony, PHI, and EAE induction. These results suggest that the encephlitogenic and antibody-combining activities reside in separate or overlapping regions of the polypeptide chain. The retention of encephalitogenic activity in relatively small pepsin peptides further suggests that the EAE determinant may be defined by a region of the polypeptide chain lacking a folded tertiary structure.

Serum α1-antitrypsin, α1-acid glycoprotein, baptoglobin, ceruloplasmin and C-reactive protein are increased, albumin, transferrin, α1-and β-lipoprotein are decreased as an early response to tissue trauma and inflammatory disease. In infection these changes are followed by a rise of γ-globulins.

Highly purified orosomucoid was exposed to periodic acid at concentrations of 0.12 and 0.60 n for 6 and 12 hours, and the oxidized products were investigated as to carbohydrate and amino acid composition, and electrophoretic and antibody-binding characteristics. Marked alterations in the properties of the native glycoprotein were produced by the periodic acid treatment. Changes in carbohydrate and amino acid composition were accompanied by a reduction in solubility, an increase in elect rophoretic mobility, a decrease in stain affinity, and a complete loss of native immunochemical specificity. The etiology and significance of these changes are discussed.

1. 1. Immunochemically homogeneous orosomucoid (α1-acid glycoprotein, α1seromucoid, seromucoide acide) was incubated with a partially purified hyaluronidase preparation, and the crude digest was subjected to gel filtration on Sephadex G-100. The three non-dialyzable fractions obtained by this procedure were analyzed by chemical, electrophoretic and immunochemical methods. 2. 2. Although they possessed essentially identical carbohydrate compositions, three fractions exhibited markedly different physical, chemical, and immunochemical properties. Fraction I retained the electrophoretic mobility of the native glycoprotein in agar gel, but had an effective molecular size∗∗ at least equal to that of 7-S globulins. It precipitated weakly with orosomucoid-specific antibody, and possessed a precipitin-inhibitory capacity only 27% that of the intact molecule. Fraction II, while appearing essentially unchanged in molecular size and electrophoretic mobility, had a markedly reduced protein stain affinity, and a precipitin-inhibitory capacity only 78% that of the native material. Fraction III had an effective molecular size similar to that of polypeptides with molecular weights between 4000 and 8000, and possessed a considerably elevated carbohydrate-to-protein ratio. In contrast to the other two fractions, it was soluble in a 5% acetic acid-70% ethanol fixative, and was neither capable of precipitating with anti-orosomucoid antibody, nor of inhibiting the latter's interaction with native glycoprotein. 3. 3. These results are presented as further evidence in support of a non-involvement of the carbohydrate moiety of the glycoprotein in its antigenicity in vitro, and may also be of value toward elucidation of its molecular structure.

Amino groups on the antibody surface (amino terminus and Lys) are very interesting conjugation targets due to their substantial quantities and selectivity toward various reactive groups. Oriented immobilization of antibodies via amino moieties on the Fc region instead of the antigen-binding fragment (Fab) is highly appreciated to conserve antigen-binding capacity. In this paper, targeting amino moieties on distinct regions, three antibody immobilization strategies were compared with the recognition ability of corresponding adsorbents. Our results demonstrate that oriented immobilization of antibodies onto heterofunctional chelate-epoxy support selectively involving Lys residues placed at the bottom of the Fc region, thus preserved the highest antigen recognition capacity (over 75% functionality). For homofunctional aldehyde support, immobilization at pH 10 demonstrates 50% remaining functionality due to the random orientation of tethered antibodies; while only 10% functionality remained when N-terminus were specifically conjugated at pH 8.5. With the rationalization of moieties density onto heterofunctional support, 2-fold recognition capacity was exhibited over randomly immobilization for antigens with higher size (β-galactosidase, 425 kDa vs. horseradish peroxidase, 40 kDa). Meanwhile, at least 97% of antigens with a varied concentration in diluted human serum were efficiently captured by the optimized chelate-epoxy support. Therefore, our antibody immobilization protocol proved the potential to be utilized as a promising candidate to capture voluminous antigens (large proteins and cells) in real samples.

3-Nitrotyrosine (3-NT) is thought to be a relevant biomarker of nitrosative stress which is associated with many inflammatory and chronic diseases. It is necessary to develop confidential method for specific and sensitive 3-NT detection. In this paper, on the basis of anti-3-NT specific antibody, we developed a label-free indirect competitive surface plasmon resonance (SPR) immunosensor and ELISA for the detection of 3-NT. Under the optimized conditions, the SPR immunosensor can obtain a linear range of 0.17–6.07 μg/mL and a limit of detection (LOD) of 0.12 μg/mL while the ELISA can reach 0.33–9.94 μg/mL and a LOD of 0.24 μg/mL.The selectivity of 3-NT was also testified by six kinds of amino acid analogues. Besides, the developed SPR immunosensor was compared thoroughly with a conventional ELISA in spiked analysis of urine samples. Good recoveries and correlation between these two methods were observed (R2 = 0.964). Therefore, it is concluded that the automated SPR platform can be applied to quantify 3-NT in biological samples with its sensitivity, accuracy, and real-timing.

Background aims Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population. Methods Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature. Results In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction. Conclusions The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters.

Enzyme-linked Immunosorbent Assays (ELISAs) techniques are widely known for the robust signal amplification with high sensitivity and specificity for the quantitation of antibodies and antigens in complex biological fluids. However, strict caution in the pre-analytical, analytical, and post-analytical phases is crucial to warrant an immunoassay quality. Since analytical errors are commonly seen in papers, this study aimed to discuss the main principles and guidelines to guarantee the specificity and sensitivity in the post-analytical phase of the ELISA method. Briefly, we highlighted the relevance of the analytical noise, as well as the limit of detection and quantitation, in the sensitivity of an immunoassay. Moreover, strategies involving the use of reverse pipetting and the spike-and-recovery test can decrease the inter-assay variance, while the linearity-of-dilution assay may protect against the prozone (or hook) effect. Addressed to researchers from different fields, we offer recommendations to limit variability and increase the usefulness of the ELISA assay in a practical way that will assist scientists to prevent future analytical errors.

The development of fast Fourier transform (FFT) algorithms enabled the sampling of billions of complex conformations and thus revolutionized protein-protein docking. FFT-based methods are now widely available and have been used in hundreds of thousands of docking calculations. Although the methods perform "soft" docking, which allows for some overlap of component proteins, the rigid body assumption clearly introduces limitations on accuracy and reliability. In addition, the method can work only with energy expressions represented by sums of correlation functions. In this paper we use a well-established protein-protein docking benchmark set to evaluate the results of these limitations by focusing on the performance of the docking server ClusPro, which implements one of the best rigid body methods. Furthermore, we explore the theoretical limits of accuracy when using established energy terms for scoring, provide comparison with flexible docking algorithms, and review the historical performance of servers in the CAPRI docking experiment.

The theory that antibody (Ab) directed against the TSH receptor (TSHR) (TSHRAb) is the causal factor of Graves' disease seems unlikely. Corticosteroids have not had a curative effect on the hyperthyroidism of Graves' disease despite their effectiveness for other autoimmune diseases. Two kinds of TSHRAb, thyroid-stimulating Ab (TSAb) and thyroid-blocking Ab (TBAb), are known as causal factors of hyperthyroidism and hypothyroidism, respectively. Previously, we reported that TSAb may be thyroid stimulating animal IgG-like hormone and TBAb may be the precursor of TSAb. In this paper we suggested that TBAb (precursor) converts to TSAb (active form) via the action of the protease, colloid antigen 2 (CA2). We speculate that the conversion of TBAb to TSAb is controlled by two factors: the protease and an anti-protease Ab. When anti-protease Ab levels are high, the patient exhibits hypothyroidism due to the increase in TBAb levels caused by neutralization of the protease. When anti-protease Ab levels are negative, the patient's hypothyroidism disappeared by the negative serum TBAb due to increased protease. An immunoglobulin G (IgG) with enzyme activity is known as an abzyme, which may be an undeveloped form. IgG with hormone activity may be likewise called an abhormone, which could also be an undeveloped form. The tumor marker CEA is a known member of the IgG supergene family. Many ancestral versions of proteins may have been produced as an IgG form. Possible participation of colloid antigen 2 and abhormone for the etiology of Graves' disease is suggested.

When a cell is infected by a virus, or becomes transformed into a malignant state, it presents clues to its disease on the outer surface of its membrane. Such clues include peptide fragments of proteins produced inside the cell; when the cell is infected by a virus, viral peptides, as well as the cell's normal peptides, are displayed on the cell's membrane as potential antigens. Infected and malignant cells also externally present special molecules that ligate NKG2D receptors on immune cells. When patrolling T lymphocytes detect the presence of both their cognate peptide antigen and NKG2D ligands on one target, they proliferate and increasingly kill other cognate target cells. The danger model of immunity recognizes NKG2D ligands as stimulators of T cell cytotoxicity, but heretofore could not explain how T cells specific to normal peptides typical of healthy host cells outside the thymus, could avoid activation by danger signals on diseased cells. The problem is that T cells specific to host-peptides are also stimulated when those peptides are by chance also displayed on diseased cells displaying NKG2D ligands. However, if T cells predicated their cytotoxicity not only on the presence of their cognate antigen found in conjunction with danger signals, but also on the absence of their cognate antigen on cells not presenting danger signals, then only T cells specific for disease antigens would become activated. Since Fas display is correlated with viral or malignant transformation, it may be a danger-signal like NKG2D ligands. T cells which encounter Fas on malignant, cognate cells, increasingly bind Fas on healthy bystander cells not displaying cognate antigens. Perhaps such healthy bystander cells provide T cells with reference-levels of danger-signals for local tissue cells, allowing T cells to select between tolerance and cytotoxic reaction to their cognate antigen, as they circulate in the periphery. This paper will analyze cytotoxicity assays that show that T cells challenge syngeneic, non-cognate bystanders with Fas ligand (FasL), and why syngeny is a requirement for danger-reference cells. Some heretofore unexplained effects of superantigens will be suggested to be due to their obstruction of reference-target detection. This paper will also suggest that established tumors often evolve a subpopulation of high-danger-signal, low tumor-antigen cells that protect the tumor against T cells; that characteristics of dendritic cells (DC) complement the danger sensing of T cells; and that DC may also use quantitatively comparative, self-referential, danger-correlation measurements to recognize transformed cells interspersed among healthy host tissue cells.

Introduction Migraine is a common neurological disease and is listed second among the most disabling health conditions worldwide. Refractory migraine (RM) is a term used to emphasize the unresponsiveness of migraine to various treatment options, encompassing both episodic refractory and chronic refractory migraine. In this paper we discuss various known and possible mechanisms of pharmacological refractoriness in RM, such as possible involvement of the gut microbiome, the blood-brain barrier, migraine genetics and various mechanisms of pharmacokinetic and pharmacodynamic tolerance. Development of medication-overuse headache as a secondary disorder following migraine is also considered. We argue that the available literature is insufficient to fully explain the mechanisms of refractoriness and we present our hypothesis. HYPOTHESIS. Refractoriness to drugs in migraine may be the result of developing anti-drug antibodies. Most migraine drugs are small molecules, which cannot elicit an immune response on their own due to their size. However, such molecules can bind to peptide carriers in their vicinity, greatly increasing their immunogenicity. A small molecule possessing this binding ability is called a hapten. Haptens form hapten-carrier complexes (HCCs), which can evoke powerful immune responses. Immune reactions to HCCs are known to be predominantly ‘drug allergies’ or type 1 drug hypersensitivity reactions’, usually resulting from IgE or non-IgE mediated mast cell degranulation. We argue that the immune reaction to HCCs can take shape in developing neutralizing anti-drug antibodies (ADA) in the form of IgG and IgA class antibodies. Since biological therapeutics, such as various monoclonal antibodies, face the issue of ADA-induced drug tolerance, HCCs, being similar in the sense that they carry peptide antigens, are of sufficient size and may be considerably immunogenic, can be responded to in a similar way by producing neutralizing ADA. Furthermore, we argue that such responses are expected to happen more frequently than is thought, due to IgG and IgA being prevalent antibodies, which utilize their neutralizing capabilities on regular basis. Finally, it is important to consider that neutralization reactions in normal immune responses are typically asymptomatic, with the only clinical expression being progressive drug tolerance. These cases may be overshadowed by the life-threatening cases of drug allergy induced anaphylaxis, possibly leading to neutralization reactions being underrecognized. Discussion This hypothesis aims to stimulate more research regarding drug resistance, and if it receives support from empirical evidence, it may help further elucidate the mechanisms underlying refractory diseases and contribute to the development of more effective treatment of many disorders.

Antigen uptake in the gastrointestinal tract may induce tolerance, lead to an immune response and also to infection. In mammals, most pathogens gain access to the host though the gastrointestinal tract, and in fish as well, this route seems to be of significant importance. The epithelial surface faces a considerable challenge, functioning both as a barrier towards the external milieu but simultaneously being the site of absorption of nutrients and fluids. The mechanisms allowing antigen uptake over the epithelial barrier play a central role for maintaining the intestinal homeostasis and regulate appropriate immune responses. Such uptake has been widely studied in mammals, but also in fish, a number of experiments have been reported, seeking to reveal cells and mechanisms involved in antigen sampling. In this paper, we review these studies in addition to addressing our current knowledge of the intestinal barrier in fish and its anatomical construction.

29 páginas, 1 figura, 1 tabla -- The definitive version is available at http://www.elsevier.com, Ticks are parasites of great medical and veterinary importance since they are vectors of numerous pathogens that affect humans, livestock and pets. Among the argasids, several species of the genus Ornithodoros transmit serious diseases such as tick-borne human relapsing fever (TBRF) and African Swine Fever (ASF). In particular, Ornithodoros erraticus is the main vector of these two diseases in the Mediterranean while O. moubata is the main vector in Africa. The presence of these Ornithodoros ticks in domestic and peridomestic environments may greatly hinder the eradication of TBRF and ASF from endemic areas. In addition, there is a constant threat of reintroduction and spreading of ASF into countries from where it has been eradicated (Spain and Portugal) or where it was never present (the Caucasus, Russia and Eastern Europe). In these countries, the presence of Ornithodoros vectors could have a tremendous impact on ASF transmission and long-term maintenance. Therefore, elimination of these ticks from at least synanthropic environments would contribute heavily to the prevention and control of the diseases they transmit. Tick control is a difficult task and although several methods for such control have been used, none of them has been fully effective against all ticks and the problems they cause. Nevertheless, immunological control using anti-tick vaccines offers an attractive alternative to the traditional use of acaricides. The aim of the present paper is to offer a brief overview of the current status in control measure development for Ornithodoros soft ticks, paying special attention to the development of vaccines against O. erraticus and O. moubata. Thus, our contribution includes an analysis of the chief attributes that the ideal antigens for an anti-tick vaccine should have, an exhaustive compilation and analysis of the scant anti-soft tick vaccine trials carried out to date using both concealed and salivary antigens and, finally, a brief description of the new reverse vaccinology approaches currently used to identify new and more effective protective tick antigens, We thank the financial support provided by the Spanish Ministry of Science and Innovation (Grants no. AGL2010-18164 and AGL2013-42745-P)

Alum (or aluminum-containing) adjuvants are key components of many vaccines currently on the market. The immuno-potentiation effect of alum adjuvants is presumably due to their interaction with antigens, leading to adsorption on the alum particle surface. Understanding the mechanism of antigen adsorption/desorption and its influencing factors could provide guidance on formulation design and ensure proper in-vivo immuno-potentiation effect. In this paper, surface adsorption of several model proteins on two types of aluminum adjuvants (Alhydrogel® and Adjuphos®) are investigated to understand the underlying adsorption mechanisms, capacities, and potential influencing factors. It was found that electrostatic interactions are the major driving force for surface adsorption of all the model proteins except ovalbumin. Alhydrogel has a significantly higher adsorption capacity than Adjuphos. Several factors significantly change the adsorption capacity of both Alhydrogel and Adjuphos, including molecular weight of protein antigens, sodium chloride, phosphate buffer, denaturing agents, and size of aluminum particles. These important factors need to be carefully considered in the design of an effective protein antigen-based vaccine.

Epitopes are immunogenic regions in antigen protein. Prediction of B-cell epitopes is critical for immunological applications. B-cell epitopes are categorized into linear and conformational. The majority of B-cell epitopes are conformational. Several machine learning methods have been proposed to identify conformational B-cell epitopes. However, the quality of these methods is not ideal. One question is whether or not the prediction of conformational B-cell epitopes can be improved by using ensemble methods. In this paper, we propose an ensemble method, which combined 12 support vector machine-based predictors, to predict the conformational B-cell epitopes, using an unbound dataset. AdaBoost and resampling methods are used to deal with an imbalanced labeled dataset. The proposed method achieves AUC of 0.642–0.672 on training dataset with 5-fold cross validation and AUC of 0.579–0.604 on test dataset. We also find some interesting results with the bound and unbound datasets. Epitopes are more accessible than non-epitopes, in bound and unbound datasets. Epitopes are also preferred in beta-turn, in bound and unbound datasets. The flexibility and polarity of epitopes are higher than non-epitopes. In a bound dataset, Asn (N), Glu (E), Gly (G), Lys (K), Ser (S), and Thr (T) are preferred in epitope regions, while Ala (A), Leu (L) and Val (V) are preferred in non-epitope regions. In the unbound dataset, Glu (E) and Lys (K) are preferred in epitope sites, while Leu (L) and Val (V) are preferred in non-epitiopes sites.

Immobilization of antibody molecules onto hydrophobic polymeric surfaces with disordered orientation is something unwanted in many applications. To overcome this drawback, controlled immunoglobulin G (IgG) immobilization onto poly(vinylidene fluoride) surface was investigated in this paper. A two-step process involving radiofrequency plasma pretreatment for polymer surface functionalization, followed by coupling reaction was developed, after which immunoglobulin G was immobilized onto the surface directly or via protein-A. IR and XPS data proved that the process is more efficient when the radiofrequency plasma pretreatment was performed using N 2 and N 2 /H 2 as discharge gases. NIR-CI, AFM and XPS surface evaluation revealed that immobilization of IgG onto N 2 /H 2 plasma-treated PVDF via grafted protein-A was achieved with an ends-on orientation, leaving available the antigen binding sites of IgG. This procedure could be a promising route for the preparation of oriented IgG assembly onto PVDF, useful in biomedical, membranes or sensors applications. QCM results showed a better antibody–antigen interaction when IgG immobilization onto PVDF substrate is mediated by protein A.

Immunoassay can be divided into two aspects, one is immobilization of antibodies for the efficient detection of corresponding antigens, and the other is immobilization of antigens to search for antibodies which work against them. In this paper, we demonstrated these two aspects of immunoassay by using the disposable fiber optic biosensors based on surface plasmon resonance (SPR) through surface decoration with half-antibody fragments, which has been scarcely ever reported to the best of our knowledge. We first fabricated the fiber optic SPR biosensor which consists of one gold film coated single-mode fiber sandwiched by two multimode fibers. Then, we decorated the fiber optic SPR biosensors with antibody fragments and antigen fragments, respectively, and compared the specific detection performances of these two kinds of sensors. After surface decoration with half-antibody fragments, the antigen-decorated fiber probe has a demonstrated sensitivity and limit of detection of 0.9771nm/(μg/mL) and 0.1 μg/mL, respectively, which improves by 10 times compared with the performance of the antibody-decorated fiber probe. Additionally, the selective detection results indicate that our proposed biosensor can be employed as a reliable antigen detector or an effective antibody filter. Our proposed sensor has the advantages of miniaturization, low cost, simple usage, label-free detection, high efficiency and sensitivity, and can effectively avoid cross-contamination caused by reuse. Given the reliable and clean detection method for immunoassay, our work should open a new window for the utilization of miniaturized fiber optic sensors in biochemical sensing.

Despite recent advancements in therapy, melanoma remains a highly lethal skin cancer. A better understanding of the genetic and epigenetic changes responsible for melanoma formation and progression could result in the development of more effective treatments. Advanced melanomas are known to exhibit widespread promoter region CpG island methylation leading to the inactivation of key tumor suppressor genes. Meta-analyses of relevant microarray data sets revealed the hematopoietic stem cell regulator gene latexin (LXN) to be commonly downregulated in approximately 50% of melanomas. The CpG island in the promoter region of LXN was almost universally hypermethylated in melanoma cell lines and tumors, and treatment of the cell lines with the demethylating drug 5-aza-2'-deoxycytidine resulted in increased LXN expression. In this paper, we demonstrate that the exogenous expression of LXN in melanoma cell lines results in a significant inhibition of tumor cell proliferation. In addition, we show that the increased expression of LXN in these lines correlates with reduction in the expression levels of stem cell transcription factors OCT4, NANOG, SOX2, KLF4, and MYCN, indicating that LXN may exert its tumor-suppressive function by altering the stem cell-like properties of melanoma cells.

In this paper, a novel method was proposed to prepare artificial antigens of gonyaulax parlaytic shellfish toxin 2 and 3 (GTX2,3). An intermediate GTX2,3-aldehyde was first synthesized by activating the NH₂ group of the 2nd and 8th amino acid residues with three different aldehydes and two artificial complete antigens GTX2,3-aldehyde-bovine serum albumin (BSA) and GTX2,3-aldehyde- keyhole limpet hemocyanin (KLH) were then prepared by cross-linking the intermediate with BSA or KLH. The successful preparation of the two complete antigens was confirmed by UV spectral scanning, HPLC, production of antibodies with titer of 1.28 × 10⁴ from mice immunized with the two complete antigens, indirect ELISA and Western-blot. In conclusion, the synthesized complete antigens have strong immunogenicity, which provides a solid foundation for preparing GTX2,3 monoclonal antibody and rapid detection kit.

The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

Understanding the influences of particle properties of antigen-adjuvant complexes on immunity is crucial in designing highly active adjuvants for new-generation of vaccines. This paper briefly revisits the current opinions on the size-dependent immunity of various adjuvant particles and then comprehensively discusses a few immunity-determining processes that are affected by the antigen-adjuvant particle properties. These include particle size, surface charge, surface hydrophilicity/lipophilicity, and antigen-adjuvant binding strength. Based on current understandings, we hypothesize that a maximum immune response occurs at a certain antigen-adjuvant particle size. This hypothesis clearly explains the paradoxical opinions on the size-dependent immunity and has also been supported by the data reported by several research groups. Finally, we further hypothesize that there is a similar relationship between any immune response and any measureable antigen-adjuvant particle property, and that there is a maximum immune response when all measureable antigen-adjuvant particle properties are optimized. We believe more attention should be paid to this issue when designing and developing effective adjuvants in future research.

Within this paper we describe the use of scanning electrochemical microscopy (SECM) to fabricate a dotted array of biotinylated polyethyleneimine which was then used to immobilise first neutravidin and then a biotinylated antibody towards a relevant antigen of interest (PSA, NTx, ciprofloxacin). These antigens were selected both for their clinical relevance but also since they display a broad range of molecular weights, to determine whether the size of the antigen used effects the sensitivity of this approach. The SECM was then used to image the binding of both complementary and non-complementary antigens in a label-free assay. Imaging of the arrays before and following exposure to various concentrations of antigen in buffer showed clear evidence for specific binding of the complementary antigens to the antibody functionalised dots. Non-specific binding was also quantified by control experiments with other antigens. This demonstrated non-specific binding across the whole of the substrate, thereby confirming that specific binding does occur between the antibody and antigen of interest at the surface of the dots. The binding of ciprofloxacin was investigated both in simple buffer solution and in a more complex media, bovine milk.

Objective: Helper T-cell epitopes (Th epitopes) are the basic units which activate helper T-cell's immune response, and they are helpful for understanding the immune mechanism and developing vaccines. Peptide and major histocompatibility complex class II (MHC-II) binding is an important prerequisite event for helper T-cell immune response, and the binding peptides are usually recognized as Th epitopes, therefore we can identify Th epitopes by predicting MHC-II binding peptides. Recently, instead of differentiating the peptides as binder or non-binder, researchers are more interested in predicting binding affinities between MHC-II molecules and peptides. Methodology: Motivated by the collective search strategy of the particle swarm optimization algorithm (PSO), a method was developed to make the direct prediction of peptide binding affinity. In our paper, PSO was utilized to search for the optimal position-specific scoring matrices (PSSM) from the experimentally derived allele-related peptides, and then the prediction models were constructed based on the matrices. Moreover, we evaluated several factors influencing the binding affinity, including peptide length and flanking residue length, and incorporated them into our models. Results: The performance of our models was evaluated on three MHC-II alleles from AntiJen database and 14 MHC-II alleles from IEDB database. When compared to the existing popular quantitative methods such as MHCPred, SVRMHC, ARB and SMM-align, our method can give out better performance in terms of correlation coefficient (r) and area under ROC curve (AUC). In addition, the results demonstrated that the performance of models was further improved by incorporating the global length information, achieving average AUC value of 0.7534 and average r value of 0.4707. Conclusions: Quantitative prediction of MHC-II binding affinity can be modeled as an optimization problem. Our PSO based method can find the optimal PSSM, which will then be used for identifying the binding cores and scoring the binding affinities of the peptides. The experiment results show that our method is promising for the prediction of MHC-II binding affinity.

Functional electrical stimulation (FES) can restore control and offset atrophy to muscles after neurological injury. However, FES has not been considered as a method for enhancing CNS regeneration. This paper demonstrates that FES dramatically enhanced progenitor cell birth in the spinal cord of rats with a chronic spinal cord injury (SCI). A complete SCI at thoracic level 8/9 was performed on 12 rats. Three weeks later, a FES device to stimulate hindlimb movement was implanted into these rats. Twelve identically-injured rats received inactive FES implants. An additional control group of uninjured rats were also examined. Ten days after FES implantation, dividing cells were marked with bromodeoxyuridine (BrdU). The ‘cell birth’ subgroup (half the animals in each group) was sacrificed immediately after completion of BrdU administration, and the ‘cell survival’ subgroup was sacrificed 7 days later. In the injured ‘cell birth’ subgroup, FES induced an 82-86 % increase in cell birth in the lumbar spinal cord. In the injured ‘cell survival’ subgroup, the increased lumbar newborn cell counts persisted. FES doubled the proportion of the newly-born cells which expressed nestin and other markers suggestive of tripotential progenitors. In uninjured rats, FES had no effect on cell birth/survival. This report suggests that controlled electrical activation of the CNS may enhance spontaneous regeneration after neurological injuries.

The adsorption of recombinant botulinum neurotoxin (BoNT) protein-derived vaccine antigens to aluminum salt adjuvants has been previously studied for the development of a trivalent vaccine against the neurotoxins (Vessely et al., in press, J Pharm Sci). The current paper describes an investigation of the stability of recombinant BoNT antigens adsorbed to aluminum salt adjuvants during storage in aqueous solution. Both chemical and physical changes occurred during storage. Phosphate groups in the buffer exchanged with hydroxyl groups on the adjuvant surface. The resulting changes in solution pH and adjuvant surface chemistry promoted more favorable electrostatic interaction between the BoNT proteins and the surface, possibly increasing the affinity of the proteins for the surface during storage. Fluorescence and UV spectroscopy suggested changes to protein structure during storage, whereas differential scanning calorimetry showed changes to thermal processes related to protein conformation and/or surface adsorption. The consequence of the chemical and physical changes to the proteins was a decrease in the ability to desorb protein from the adjuvant surface during storage. Overall, the results of this study emphasize the utility of a thorough characterization of the interactions between protein antigens and aluminum salt adjuvants.

Apurinic/apyrimidinic endonuclease-1/Redox factor-1, a multifunctional DNA base excision repair and redox regulation enzyme, plays an important role in oxidative signalling, transcription factor regulation, and cell cycle control. Recently, we have demonstrated that following the triggering of CD40 on B cells, APE/Ref-1 translocates from the cytoplasm to the nucleus and regulates the activity of B cell-specific transcription factors. In the present paper we investigate whether APE/Ref-1 plays a role in controlling CD40-mediated B cell proliferation too. We demonstrate a concurrent increase in proliferation and decrease in apoptosis of primary mouse B cells activated by CD40 cross-linking and transfected with functional APE/Ref-1 antisense oligonucleotide. Moreover, we provide evidence that a redox-mediated signalling mechanism is involved in this process and we propose that APE/Ref-1, controlling the intracellular redox state, may also affect the cell cycle by inducing nucleus-cytoplasm redistribution of p21. Together, these findings suggest that APE/Ref-1 could act as a negative regulator in an adaptive response to elevated ROS levels following CD40 cross-linking. Considering the important role of ROS and APE/Ref-1 in CD40-mediated B cell proliferation, our data will contribute to understand the mechanisms of tumor escape and suggest APE/Ref-1 as a novel target for tumor therapeutic approaches.

In this paper, an immune-inspired model, named innate and adaptive artificial immune system (IA-AIS) is proposed and applied to the problem of identification of unsolicited bulk e-mail messages (SPAM). It integrates entities analogous to macrophages, B and T lymphocytes, modeling both the innate and the adaptive immune systems. An implementation of the algorithm was capable of identifying more than 99% of legitimate or SPAM messages in particular parameter configurations. It was compared to an optimized version of the naive Bayes classifier, which has been attained extremely high correct classification rates. It has been concluded that IA-AIS has a greater ability to identify SPAM messages, although the identification of legitimate messages is not as high as that of the implemented naive Bayes classifier.

Cattle tick control remains a serious problem in cattle farms worldwide, due to the limited success achieved with chemicals. Although the use of vaccines for tick control may open possibilities for an integrated approach, the search for other protective antigens is still necessary to improve control. Boophilus microplus is a rich source of BmTIs, serine proteinase inhibitors that are present throughout the tick's lifecycle. The present paper reports a pen trial conducted to evaluate the performance of a synthetic BmTI N-terminal fragment as antigen against the tick in bovines. The trial was conducted with two groups of eight crossbred cattle under controlled infestation: one group was vaccinated with BmTI peptide using saponin as adjuvant and the other was kept as control. Challenge was performed with 15,000 larvae. The specific IgG response was measured by ELISA, with successful outcomes for vaccinated animals. Vaccination resulted in an 18.4% level of efficacy when compared with the control group.

The ideal immobilization methods that are suitable for binding immuno-active materials with high efficiency onto the sensing surface are the key target to pursue in the current biosensor design. In this paper, a new hybrid material formed by assembling gold nanoparticles (GNP) onto nano-sized hydroxyapatite (HA) has been employed for the interface design of piezoelectric immunosensor, on which the antibodies were bound. The detection performances of the resulting immunosensor were investigated by use of the antibody-antigen model system of alpha-Fetoprotein (AFP), an important indicator in the diagnosis of clinical cancers. The hybrid material was characterized by the UV-vis spectroscopy, the SEM and TEM measurements. The frequency and electrochemical impedance responses characteristics for the processes of immobilization and immunoreaction of anchored anti-AFP antibodies were studied in detail. The immunoresponse of the proposed immunosensor was compared with those antibodies immobilized by using HA or GNP alone. It was found that the developed sensing interface has some advantages such as the activation-free immobilization and the high antigen-binding activities of antibodies. The as-prepared immunosensor can allow for the determination of AFP in the concentration range of 15.3-600.0ngml(-1). Such an interface design with the nano-sized hybrid materials should be tailored as a new alternative used for biosensor design.

The evaluation of potential adverse effects of pharmaceuticals on the immune system is part of the standard drug development procedures and needs to be available prior to the start of phase III clinical trials. Although the histopathological assessment of lymphoid organs/tissues is considered fundamental for the identification and characterization of immunotoxic reactions, additional investigations are now recommended by the European guidelines for repeated-dose toxicity testing of medicinal products in order to achieve an accurate assessment of immune system functionality with regard to immunomodulation. In the present paper, we describe and discuss a study design which permits the investigation of the immune function in a 4-week study in rats following immunization by subcutaneous administration of the T-dependent antigen Keyhole Limpet Hemocynin (KLH). This includes assessment of hematology parameters, titration of KLH-specific antibodies in serum, lymphocyte immunophenotyping in blood, thymus, spleen and lymph nodes, macroscopic pathology and histopathological evaluation of the lymphatic organs/tissues and of the injection sites.